Because of the potential bioterrorism threat, the development of safe and effective vaccines against the infectious microorganisms on the select agents list is a national and worldwide goal. In some cases there are vaccines in existence but they are considered unsafe for widespread use on large populations. For example the live attenuated smallpox and F. tularensis vaccines are used only under restricted circumstances for laboratory workers, "first responders", or for other groups at high risk of potential exposure. Protein subunit vaccines and DMA vaccines are safer alternatives compared with live attenuated or killed microorganism vaccines. Recent progress in our laboratory using whole proteome microarray scanning has lead to discovery of a number of potential candidate antigens for use in subunit vaccines against smallpox and tularemia. However, proteins require adjuvants in order to improve vaccine potency and protective efficacy. Furthermore, the potency of DNA vaccines can be increased by boosting with recombinant viruses or proteins, and the "Prime-Boost" approach is becoming accepted with some enthusiasm as an effective way to improve the immune responses against recombinant antigens. However, systematic studies designed to investigate the benefits of DNA prime together with adjuvanted protein boost are lacking. Here we will investigate the DNA vaccine prime and recombinant protein boost using 4 novel adjuvants in the protein boost. In Specific Aim 1, we will focus on using two model antigens, one derived from vaccinia virus (the smallpox vaccine) and a second from F. tularensis. The wild-type F. tularensis gene will be codon optimized for expressions in mammalian cells so that they will be effective as DNA vaccines. Each protein will also be expressed in bacteria, purified and formulated in each adjuvant for use in the boost. Assays will be established to determined serum levels of all antibody types and IgG subtypes against each antigen, and a cytokine panel will be assayed on antigen restimulated spleen cells in order to distinguish TH1 and TH2 responses. In Specific Aim 2, all four of the adjuvants will be evaluated in the DNA vaccine prime & protein boost protocol, and antigen specific humoral and cellular immune responses will be quantified. In Specific Aim 3 the best performing vaccine configurations will be tested in live infection models against vaccinia and F. tularensis using combinations of antigens identified in our previous vaccine discovery effort. Successful completion of the studies described in this application will set a stage for our subsequent plan to submit a Phase 2 application focusing on studies that will eventually lead to clinical trials of safer and effective subunit vaccines against smallpox and F tularensis.